技术资料

A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here,human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ωtextperiodcenteredcm(2). By assessing the permeabilities of several known drugs,a benchmarking system to evaluate brain permeability of drugs was established. Furthermore,relationships between TEER and permeability to both small and large molecules were established,demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype,and the benchmarks established here are useful for assessing functionality of other in vitro BBB models. View Publication

Myocardial ischemia-reperfusion (I/R) injury is unavoidable during cardioplegic arrest and open-heart surgery. Danshen is one of the most popular traditional herbal medicines in China,which has entered the Food and Drug Administration-approved phase III clinical trial. This study was aimed to develop a human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) model to mimic I/R injury and evaluate the cardioprotective effect of regular cardioplegic solution with Danshen. hiPSC-CMs were cultured with the crystalloid cardioplegic solution (Thomas group) and Thomas solution with 2 or 10 µg/mL Danshen (Thomas plus Danshen groups). The cells under normoxic culture condition served as baseline group. Then,the cells were placed in a modular incubator chamber. After 45 min hypoxia and 3 h reoxygenation,hiPSC-CMs subjected to hypoxia/reoxygenation resulted in a sharp increase of reactive oxygen species (ROS) content in Thomas group versus baseline group. Compared with the Thomas group,ROS accumulation was significant suppressed in Thomas plus Danshen groups,which might result from elevating the content of glutathione and enhanced activities of superoxide dismutase and glutathione peroxidase. The enhanced L-type Ca(2+) current in hiPSC-CMs after I/R injury was also significantly decreased by Danshen,and meanwhile intracellular Ca(2+) level was reduced and calcium overload was suppressed. Thomas plus Danshen groups also presented less irregular transients and lower apoptosis rates. As a result,Danshen could improve antioxidant and calcium handling in cardiomyocytes during I/R and lead to reduced arrhythmia events and apoptosis rates. hiPSC-CMs model offered a platform for the future translational study of the cardioplegia. View Publication

Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children,with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue. Pathogenic mtDNA mutations are relatively common,with an estimated 778 affected children born each year in the United States. Mitochondrial replacement therapies or techniques (MRT) circumventing mother-to-child mtDNA disease transmission involve replacement of oocyte maternal mtDNA. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother's oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer,resulting in embryos containing<99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However,some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor-to-maternal mtDNA interactions,it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition,some haplotypes confer proliferative and growth advantages to cells. Hence,we propose a matching paradigm for selecting compatible donor mtDNA for MRT. View Publication

Telomere length maintenance ensures self-renewal of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs); however,the mechanisms governing telomere length homeostasis in these cell types are unclear. Here,we report that telomere length is determined by the balance between telomere elongation,which is mediated by telomerase,and telomere trimming,which is controlled by XRCC3 and Nbs1,homologous recombination proteins that generate single-stranded C-rich telomeric DNA and double-stranded telomeric circular DNA (T-circles),respectively. We found that reprogramming of differentiated cells induces T-circle and single-stranded C-rich telomeric DNA accumulation,indicating the activation of telomere trimming pathways that compensate telomerase-dependent telomere elongation in hiPSCs. Excessive telomere elongation compromises telomere stability and promotes the formation of partially single-stranded telomeric DNA circles (C-circles) in hESCs,suggesting heightened sensitivity of stem cells to replication stress at overly long telomeres. Thus,tight control of telomere length homeostasis is essential to maintain telomere stability in hESCs. View Publication

Diverse Ab effector functions mediated by the Fc domain have been commonly associated with reduced risk of infection in a growing number of nonhuman primate and human clinical studies. This study evaluated the anti-HIV Ab effector activities in polyclonal serum samples from HIV-infected donors,VAX004 vaccine recipients,and healthy HIV-negative subjects using a variety of primary and cell line-based assays,including Ab-dependent cellular cytotoxicity (ADCC),Ab-dependent cell-mediated viral inhibition,and Ab-dependent cellular phagocytosis. Additional assay characterization was performed with a panel of Fc-engineered variants of mAb b12. The goal of this study was to characterize different effector functions in the study samples and identify assays that might most comprehensively and dependably capture Fc-mediated Ab functions mediated by different effector cell types and against different viral targets. Deployment of such assays may facilitate assessment of functionally unique humoral responses and contribute to identification of correlates of protection with potential mechanistic significance in future HIV vaccine studies. Multivariate and correlative comparisons identified a set of Ab-dependent cell-mediated viral inhibition and phagocytosis assays that captured different Ab activities and were distinct from a group of ADCC assays that showed a more similar response profile across polyclonal serum samples. The activities of a panel of b12 monoclonal Fc variants further identified distinctions among the ADCC assays. These results reveal the natural diversity of Fc-mediated Ab effector responses among vaccine recipients in the VAX004 trial and in HIV-infected subjects,and they point to the potential importance of polyfunctional Ab responses. View Publication

We have compared the transcriptomic profiles of human induced pluripotent stem cells after their differentiation in hepatocytes like cells in plates and microfluidic biochips. The biochips provided a 3D and dynamic support during the cell differentiation when compared to the 2D static cultures in plates. The microarray have demonstrated the up regulation of important pathway related to liver development and maturation during the culture in biochips. Furthermore,the results of the transcriptomic profile,coupled with immunostaining,and RTqPCR analysis have shown typical biomarkers illustrating the presence of responders of biliary like cells,hepatocytes like cells,and endothelial like cells. However,the overall tissue still presented characteristic of immature and foetal patterns. Nevertheless,the biochip culture provided a specific micro-environment in which a complex multicellular differentiation toward liver could be oriented. View Publication

The cytogenetic analysis of plasma cell myeloma (PCM) allows stratification of patients so that prognosis may be determined and appropriate therapeutic options can be discussed. Owing to the patchy nature of the disease in the bone marrow (BM),the low proliferative activity of plasma cells and the cryptic nature of some PCM-associated cytogenetic changes,karyotypic analysis in this disease should be augmented with targeted interphase fluorescence in situ hybridization (FISH). Immunofluorescent revelation of cytoplasmic immunoglobulin light chains,together with interphase FISH (cIg-FISH),allows the identification of plasma cells within a sample so that they may be scored preferentially. This is particularly useful in situations where there are only a small percentage of plasma cells in a sample. Where an underlying myeloid disease is suspected the cIg-FISH-negative cells can be scored separately. Two methods are provided in this chapter: the technique for cIg-FISH in fresh PCM BM samples and a procedure for use in fixed cytogenetics preparations. View Publication

Human pluripotent stem cells (hPSCs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and requires experience in hPSC culture and differentiation techniques. Building on lessons taken from early development,this monolayer-directed differentiation protocol uses different concentrations of activin A and bone morphogenetic protein 4 (BMP4) to polarize cells into mesodermal subtypes that reflect mid-primitive-streak cardiogenic mesoderm and posterior-primitive-streak hemogenic mesoderm. This differentiation platform provides a basis for generating distinct cardiovascular progenitor populations that enable the derivation of cardiomyocytes and functionally distinct endothelial cell (EC) subtypes from cardiogenic versus hemogenic mesoderm with high efficiency without cell sorting. ECs derived from cardiogenic and hemogenic mesoderm can be matured into textgreater90% CD31(+)/VE-cadherin(+) definitive ECs. To test the functionality of ECs at different stages of differentiation,we provide methods for assaying the blood-forming potential and de novo lumen-forming activity of ECs. To our knowledge,this is the first protocol that provides a common platform for directed differentiation of cardiomyocytes and endothelial subtypes from hPSCs. This protocol yields endothelial differentiation efficiencies exceeding those of previously published protocols. Derivation of these cell types is a critical step toward understanding the basis of disease and generating cells with therapeutic potential. View Publication

To develop a purification strategy for isolating the most primitive hematopoietic stem cells present in normal human marrow we have combined cell separation techniques with an assay for cells that initiate sustained hematopoiesis in vitro in the presence of irradiated human marrow adherent cells. These feeders" were established by subculturing 2- to 6-week-old primary long-term marrow culture adherent layers at a density of 3 x 10(4) irradiated cells per square centimeter. Test "long-term culture (LTC)-initiating cells" were plated on top of the feeders and the cocultures then maintained as standard long-term marrow cultures with half-media changes and removal of half of the nonadherent cells each week. The total number of myeloid� View Publication

Human embryonic stem cells (hESCs) are used as platforms for disease study,drug screening and cell-based therapy. To facilitate these applications,it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However,the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However,certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site,probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein,LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs. View Publication

TRAF1 is a signaling adaptor known for its role in tumor necrosis factor receptor-induced cell survival. Here we show that monocytes from healthy human subjects with a rheumatoid arthritis-associated single-nucleotide polymorphism (SNP) in the TRAF1 gene express less TRAF1 protein but greater amounts of inflammatory cytokines in response to lipopolysaccharide (LPS). The TRAF1 MATH domain binds directly to three components of the linear ubiquitination (LUBAC) complex,SHARPIN,HOIP and HOIL-1,to interfere with the recruitment and linear ubiquitination of NEMO. This results in decreased NF-κB activation and cytokine production,independently of tumor necrosis factor. Consistent with this,Traf1(-/-) mice show increased susceptibility to LPS-induced septic shock. These findings reveal an unexpected role for TRAF1 in negatively regulating Toll-like receptor signaling,providing a mechanistic explanation for the increased inflammation seen with a disease-associated TRAF1 SNP. View Publication

Human NK cell deficiencies are rare yet result in severe and often fatal disease,particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells,and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8,which encodes an interferon regulatory factor,as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells,and this impairment in terminal maturation was also observed in Irf8-/-,but not Irf8+/-,mice. We then determined that impaired maturation was NK cell intrinsic,and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together,these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease,thereby emphasizing a critical role for NK cells in human antiviral defense. View Publication